The yeast two-hybrid (Y2H) assay is a method for detecting protein-protein interactions using a genetic system. The technique may be used for charting protein interactions, and hence, identifying potential partners in genetic pathways. The assay is sensitive and yields the DNA sequences encoding proteins that interact. In a typical two-hybrid assay, a known protein that forms part of a DNA-binding domain hybrid is assayed against a library of all possible proteins present as transcriptional activation domain hybrids. Some two hybrid approaches rely on interaction mating. In this method, the protein fused to the DNA-binding domain and the protein fused to the activation domain are expressed in two different haploid yeast strains of opposite mating type and the strains are mated to determine if the two proteins interact. When haploid yeast strains of opposite mating type come into contact, mating occurs and results in fusion of the two haploids to form a diploid yeast strain. An interaction can thus be determined by measuring activation of a two-hybrid reporter gene in the dipoid strain.
WO 94/10300 and U.S. Pat. No. 5,283,173 describe methods for detecting the interaction between proteins using reconstitution of the activity of a transcriptional activator. This reconstitution makes use of chimeric genes which express hybrid proteins. The first hybrid contains the DNA-binding domain of a transcriptional activator fused to a known protein (the "bait"), with the DNA binding domain DNA binding element placed upstream of a reporter gene. "Prey" proteins are cloned as either random sequences or cDNAs and are fused to the amino- or carboxy-terminus of a transcription activation domain. If the bait and prey proteins are able to interact, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription, which can be detected by the activity of a reporter gene that contains a binding site for the DNA-binding domain.
The disadvantages of these techniques is that irrelevant interactions with yeast proteins are generated. These include false-positive interactions that are unlikely to be found in living cells, and false-negative interactions, that is, those interactions that would otherwise be detected but are not. The techniques as disclosed in WO 94/10300 and U.S. Pat. No. 5,283,173 require the use of mating in solid medium which is cumbersome, labor-intensive, and does not preserve diploid cells for further analysis.
We have developed the mating strategy of the yeast two-hybrid assay into an automated format which allows many bait proteins to be processed. The format uses an arraying means, for example, microtiter plates and liquid mass-mating of a subset of a large, complex library. By tracking positive interactions in the library, we have also developed a method to create a functionally-subtracted library, that is, one that can be made devoid of a scorable phenotype. For example, our method allows for the determination of detection of hybrids that react promiscuously with many targets, such as heat shock proteins, and their elimination from any future considerations.